Hemostatic composition containing recombinant human clotting factors, and method of producing

Abstract

This invention relates to a wholly recombinant four factor hemostatic complex concentrate (4F-PCC) composition, human liver cell-produced product. Methods of producing a hemostatic composition using a hepatocyte cell line and a CRISPR/Cas9 gene activation multiplexing, which allows for simultaneous and high level expression of the FII, FVII, FIX, and FX clotting factors. The resultant product is a recombinant resuscitation solution to aid in mitigation of coagulopathy, reversal of coagulopathic states, and fluid resuscitation.

1 . A method of producing a recombinant human clotting factors FII, FVII, FIX and FX, comprising the step of:
 expressing DNA for each of human clotting factors FII, FVII, FIX and FX and encoding the respective gene products in an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Synergistic Activation Module (SAM) system in a human hepatocyte cell, comprising one or more vectors comprising   (a) a first regulatory element operable in the human hepatocyte cell operably linked to a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FII DNA sequence;
 a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FVII DNA sequence; 
 a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FIX DNA sequence; and 
 a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FX DNA sequence; and 
   (b) a second regulatory element operable in the human hepatocyte cell operably linked to a nucleotide sequence encoding a synergistic activation module (SAM), which comprises
 a nucleotide sequence encoding catalytically inactive Type-II Cas9 protein engineered to bind but not cleave DNA a DNA/RNA complex, and 
 transcriptional activation domains, and aptamers capable to form RNA aptamer stem loops into the respective guide RNAs and which specifically bind/hybridize to the respective human clotting factor,
 whereby each guide RNA targets and hybridizes to the appropriate human clotting factor FII, FVII, FIX or FX sequence, whereby expression of gene products for human clotting factor FII, FVII, FIX and FX is altered; and, wherein the Cas9 protein and the guide RNAs do not naturally occur together. 
 
   
     
     
         2 . A method for producing a recombinant human clotting factors FII, FVII, FIX and FX, comprising the steps of:
 (a) in a first human hepatocyte cell, expressing DNA for human clotting factor FII and encoding human clotting factor FII gene product in an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Synergistic Activation Module (SAM) system, comprising one or more vectors comprising
 a first regulatory element operable in the first human hepatocyte cell operably linked to a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FII DNA sequence; 
 a second regulatory element operable in the first human hepatocyte cell operably linked to a nucleotide sequence encoding a synergistic activation module (SAM), which comprises a nucleotide sequence encoding catalytically inactive Type-II Cas9 protein engineered to bind but not cleave DNA a DNA/RNA complex, and transcriptional activation domains, optionally comprising transcriptional activation subunits, and an aptamer capable to form an RNA aptamer stem loop into the guide RNA,
 whereby the guide RNA targets and hybridizes to the human clotting factor FIX sequence, whereby expression of gene product for human clotting factor FIX is altered; 
 
   (b) in a second human hepatocyte cell, expressing DNA for human clotting factor FVII and encoding human clotting factor FVII gene product in an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Synergistic Activation Module (SAM) system, comprising one or more vectors comprising
 a third regulatory element operable in the second human hepatocyte cell operably linked to a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FVII DNA sequence; 
 a fourth regulatory element operable in the second human hepatocyte cell operably linked to a nucleotide sequence encoding a synergistic activation module (SAM), which comprises a nucleotide sequence encoding catalytically inactive Type-II Cas9 protein engineered to bind but not cleave DNA a DNA/RNA complex, and transcriptional activation domains, optionally comprising transcriptional activation subunits, and an aptamer capable to form an RNA aptamer stem loop into the guide RNA,
 whereby the guide RNA targets and hybridizes to the human clotting factor FVII sequence, whereby expression of gene product for human clotting factor FVII is altered; 
 
   (c) in a third human hepatocyte cell, expressing DNA for human clotting factor FIX and encoding human clotting factor FIX gene product in an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Synergistic Activation Module (SAM) system, comprising one or more vectors comprising
 a fifth regulatory element operable in the third human hepatocyte cell operably linked to a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FIX DNA sequence; 
 a sixth regulatory element operable in the third human hepatocyte cell operably linked to a nucleotide sequence encoding a synergistic activation module (SAM), which comprises a nucleotide sequence encoding catalytically inactive Type-II Cas9 protein engineered to bind but not cleave DNA a DNA/RNA complex, and transcriptional activation domains, optionally comprising transcriptional activation subunits, and an aptamer capable to form an RNA aptamer stem loop into the guide RNA,
 whereby the guide RNA targets and hybridizes to the human clotting factor FIX sequence, 
 whereby expression of gene product for human clotting factor FIX is altered; 
 
   (d) in a fourth human hepatocyte cell, expressing DNA for human clotting factor FX and encoding human clotting factor FX gene product in an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Synergistic Activation Module (SAM) system, comprising one or more vectors comprising
 a seventh regulatory element operable in the fourth human hepatocyte cell operably linked to a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FX DNA sequence; 
 an eighth regulatory element operable in the fourth human hepatocyte cell operably linked to a nucleotide sequence encoding a synergistic activation module (SAM), which comprises a nucleotide sequence encoding catalytically inactive Type-II Cas9 protein engineered to bind but not cleave DNA a DNA/RNA complex, and transcriptional activation domains, optionally comprising transcriptional activation subunits, and an aptamer capable to form an RNA aptamer stem loop into the guide RNA,
 whereby the guide RNA targets and hybridizes to the human clotting factor FX sequence, 
 whereby expression of gene product for human clotting factor FX is altered; and, wherein none of the Cas9 proteins and the guide RNAs naturally occur together; and 
 
   (e) combining the gene products of a)-d) in a composition.   
     
     
         3 . A method of producing a recombinant human clotting factor comprising the step of:
 expressing DNA for a human clotting factor selected from FII, FVII, FIX, FX, or a combination thereof and encoding the respective gene products in an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Synergistic Activation Module (SAM) system in a human hepatocyte cell, comprising one or more vectors comprising   (a) a first regulatory element operable in the human hepatocyte cell operably linked to
 a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FII DNA sequence; 
 a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FVII DNA sequence; 
 a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FIX DNA sequence; 
 a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with human clotting factor FX DNA sequence; or 
 a combination thereof; 
   (b) a second regulatory element operable in the human hepatocyte cell operably linked to
 a nucleotide sequence encoding a synergistic activation module (SAM), which comprises 
 a nucleotide sequence encoding catalytically inactive Type-II Cas9 protein engineered to bind but not cleave DNA a DNA/RNA complex, and 
 transcriptional activation domains, and aptamers capable to form RNA aptamer stem loops into the respective guide RNAs and which specifically bind/hybridize to the respective human clotting factor,
 whereby each guide RNA targets and hybridizes to the appropriate human clotting factor FII, FVII, FIX, FX sequence, or a combination thereof, 
 whereby expression of gene products for human clotting factor FII, FVII, FIX, FX, or a combination thereof is increased. 
 
   
     
     
         4 . The method of any one of  claims 1-3 , wherein the method further comprises harvesting the human clotting factor produced, and optionally, combining into a composition. 
     
     
         5 . The method of any one of  claims 1-4 , wherein the Cas9 protein and the guide RNAs do not naturally occur together. 
     
     
         6 . The method of any one of  claims 1-5 , wherein the CRISPR-Cas system guide RNA that hybridizes with human clotting factor FII DNA sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 8-16. 
     
     
         7 . The method of any one of  claims 1-5 , wherein the CRISPR-Cas system guide RNA that hybridizes with human clotting factor FVII DNA sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 17-21. 
     
     
         8 . The method of any one of  claims 1-5 , wherein the CRISPR-Cas system guide RNA that hybridizes with human clotting factor FIX DNA sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 22-28. 
     
     
         9 . The method of any one of  claims 1-5 , wherein the CRISPR-Cas system guide RNA that hybridizes with human clotting factor FX DNA sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 29-38. 
     
     
         10 . The method of any one of  claims 1-9 , wherein the aptamer stem loop is a MS2 aptamer. 
     
     
         11 . The method of  claim 10 , wherein the MS2 aptamer comprises the nucleic acid sequence of SEQ ID NO: 1. 
     
     
         12 . The method of any one of  claims 1-9 , wherein the aptamer stem loop is a Lambda Nut-L aptamer. 
     
     
         13 . The method of  claim 12 , wherein the Lambda Nut-L aptamer comprises the nucleic acid sequence of SEQ ID NO: 2. 
     
     
         14 . The method of any one of  claims 1-9 , wherein the aptamer stem loop is a Lambda Nut-R aptamer. 
     
     
         15 . The method of  claim 14 , wherein the Lambda Nut-R aptamer comprises the nucleic acid sequence of SEQ ID NO: 3. 
     
     
         16 . The method of any one of  claims 1-9 , wherein the aptamer stem loop is a Ob aptamer. 
     
     
         17 . The method of  claim 16 , wherein the Ob aptamer comprises the nucleic acid sequence of SEQ ID NO: 4. 
     
     
         18 . The method of any one of  claims 1-9 , wherein the aptamer stem loop is a BIV TAR aptamer. 
     
     
         19 . The method of  claim 18 , wherein the BIV TAR aptamer comprises the nucleic acid sequence of SEQ ID NO: 5. 
     
     
         20 . The method of any one of  claims 1-9 , wherein the aptamer stem loop is a STNV aptamer. 
     
     
         21 . The method of  claim 20 , wherein the STNV aptamer comprises the nucleic acid sequence of SEQ ID NO: 6. 
     
     
         22 . The method of any one of  claims 1-9 , wherein the aptamer stem loop is a PP7 aptamer. 
     
     
         23 . The method of  claim 22 , wherein the PP7 aptamer comprises the nucleic acid sequence of SEQ ID NO: 7. 
     
     
         24 . A hemostatic composition comprising recombinant human clotting factors FII, FVII, FIX and FX made by the method of any one of  claims 1-23 . 
     
     
         25 . A human plasma alternative comprising the hemostatic composition of  claim 24 . 
     
     
         26 . A method for producing a recombinant human fibrinogen comprising the steps of:
 in a human cell, expressing DNA for human fibrinogen and encoding human clotting factor fibrinogen gene product in an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Synergistic Activation Module (SAM) system, comprising one or more vectors comprising   (a) a first regulatory element operable in the human cell operably linked to a nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with fibrinogen DNA sequence domains,
 wherein the fibrinogen DNA sequence domains comprises alpha, beta, and gamma domains; 
   (b) a second regulatory element operable in the human cell operably linked to a nucleotide sequence encoding a synergistic activation module (SAM), which comprises a nucleotide sequence encoding catalytically inactive Type-II Cas9 protein engineered to bind but not cleave DNA a DNA/RNA complex, and transcriptional activation domains, optionally comprising transcriptional activation subunits, and an aptamer capable to form an RNA aptamer stem loop into the guide RNA,
 wherein the guide RNA targets and hybridizes to the human fibrinogen DNA sequence domains, 
 wherein expression of gene product for human fibrinogen is increased. 
   
     
     
         27 . The method of  claim 26 , wherein the human cell is a HEK293 cell or a hepatocyte cell. 
     
     
         28 . The method of  claim 26 or 27 , wherein the CRISPR-Cas system guide RNA that hybridizes with human fibrinogen DNA sequence domain comprises the nucleic acid sequence of any one of SEQ ID NOs: 39-47 and binds the alpha domain. 
     
     
         29 . The method of any one of  claims 26-28 , wherein the CRISPR-Cas system guide RNA that hybridizes with human fibrinogen DNA sequence domain comprises the nucleic acid sequence of any one of SEQ ID NOs: 48-55 and binds the beta domain. 
     
     
         30 . The method of any one of  claims 26-29 , wherein the CRISPR-Cas system guide RNA that hybridizes with human fibrinogen DNA sequence domain comprises the nucleic acid sequence of any one of SEQ ID NOs: 56-63 and binds the gamma domain. 
     
     
         31 . A hemostatic composition comprising recombinant fibrinogen made by the method of any one of  claims 26-30 . 
     
     
         32 . A human plasma alternative comprising the hemostatic composition of  claim 31 . 
     
     
         33 . An isolated nucleic acid sequence comprising any one of the nucleic acid sequences of SEQ ID NO: 1-67. 
     
     
         34 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 1. 
     
     
         35 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 2 or 3. 
     
     
         36 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 4. 
     
     
         37 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 5. 
     
     
         38 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 6. 
     
     
         39 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 7. 
     
     
         40 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 8-16. 
     
     
         41 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 17-21. 
     
     
         42 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 22-28. 
     
     
         43 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 29-38. 
     
     
         44 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 39-47. 
     
     
         45 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 48-55. 
     
     
         46 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 56-63. 
     
     
         47 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NO: 64. 
     
     
         48 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NO: 65. 
     
     
         49 . The isolated nucleic acid of  claim 33 , wherein the nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NO: 66 or 67. 
     
     
         50 . An isolated vector comprising the isolated nucleic acid of any one of  claims 33-49 . 
     
     
         51 . An isolated vector comprising a plurality of nucleic acids sequence comprising the nucleic acid sequences of SEQ ID NO: 1-67. 
     
     
         52 . An isolated vector comprising a plurality of nucleic acids sequence comprising the nucleic acid sequences of SEQ ID NO: 8-38. 
     
     
         53 . The vector of  claim 52 , wherein the vector comprises
 any one of nucleic acid sequences of SEQ ID NOs: 8-16;   any one of nucleic acid sequences of SEQ ID NOs: 17-21;   any one of nucleic acid sequences of SEQ ID NOs: 22-28; and   any one of nucleic acid sequences of SEQ ID NOs: 29-38.   
     
     
         54 . An isolated vector comprising a plurality of nucleic acids sequence comprising the nucleic acid sequences of SEQ ID NO: 39-63. 
     
     
         55 . The vector of  claim 54 , wherein the vector comprises
 any one of nucleic acid sequences of SEQ ID NOs: 39-47;   any one of nucleic acid sequences of SEQ ID NOs: 48-55; and   any one of nucleic acid sequences of SEQ ID NOs: 56-63.   
     
     
         56 . An isolated host cell comprising the vector of any one of  claims 50-55 . 
     
     
         57 . A method for mitigating coagulopathy in a patient comprising administering a therapeutically effective amount of the hepastatic composition of  claim 24 . 
     
     
         58 . A method for mitigating coagulopathy in a patient comprising administering a therapeutically effective amount of the hepastatic composition of  claim 31 . 
     
     
         59 . A method for treating coagulopathy in a patient comprising administering a therapeutically effective amount of the hepastatic composition of  claim 24 . 
     
     
         60 . A method for treating coagulopathy in a patient comprising administering a therapeutically effective amount of the hepastatic composition of  claim 31 . 
     
     
         61 . A method for controlling or mitigating hemorrhage in a patient comprising administering a therapeutically effective amount of the hepastatic composition of  claim 24 . 
     
     
         62 . A method for controlling or mitigating hemorrhage in a patient comprising administering a therapeutically effective amount of the hepastatic composition of  claim 31 . 
     
     
         63 . A method for treating a hemorrhage in a patient comprising administering a therapeutically effective amount of the hepastatic composition of  claim 24 . 
     
     
         64 . A method for treating a hemorrhage in a patient comprising administering a therapeutically effective amount of the hepastatic composition of  claim 31 . 
     
     
         65 . A method for promoting fluid resuscitation in a patient comprising administering a therapeutically effective amount of the hepastatic composition of  claim 24 . 
     
     
         66 . A method for promoting fluid resuscitation in a patient comprising administering a therapeutically effective amount of the hepastatic composition of  claim 31 .